PL240714B1 - Method of conducing in vitro chondrocyte cell culture to obtain material for treatment of articular cartilage defects - Google Patents

Description

PL 240 714 B1

Description of the invention

The invention generally relates to obtaining tissue as a material for the treatment of any damage to cartilage, especially as a result of traumatic cartilage damage, degenerative or degenerative cartilage damage. More particularly, the invention relates to a method of carrying out an in vitro chondrocyte cell culture to obtain a material for the treatment of articular cartilage defects.

Osteoarthritis (OA), or osteoarthritis, is characterized by joint pain and joint dysfunction caused by a progressive and irreversible loss of joint cartilage. It is the fifth largest cause of disability in the United States population, after diseases of the cardiovascular, cerebrovascular and respiratory systems. Age is the major risk factor for developing OA. There are also additional factors such as obesity, injuries of the joint area, intense physical activity, e.g. competitive sports.

The number of diagnosed OA cases in many countries is still growing, e.g. in the USA in 1995-2005 there was an increase of about 6 million (Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States Part II Arthritis Rheum 2008; 58 (1): 26-35; Lawrence RC, Helmick CG, Arnett FC, et al. Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum. 1998; 41: 778799). At the same time, there is an increase in the wider population of people suffering from degenerative and rheumatic diseases. The number of diagnosed cases of degeneration in America is expected to reach 67 million, or 25% of the adult population by 2030, of which 25 million, or 9.3% of the adult population, is expected to experience reduced mobility (Hootman JM, Helmick CG. Projections of U.S. prevalence of arthritis and associated activity limitations. Arthritis Rheum. 2006; 54: 226-229). The result of these trends is an increase in the level of negative consequences in terms of quality of life, as well as a huge increase in the costs of health care systems.

Almost every patient treated surgically due to extensive degenerative changes in joints, especially in the knee, hip, shoulder and ankle joints, in the period preceding the development of generalized joint changes goes through a period in which the destruction of cartilage articular surfaces is limited to a single focus. It is the source of the degenerative-degenerative process slowly "spreading" over the pond. Along with the improvement of the patient's access to highly specialized diagnostic tools, such as MRI and arthroscopy, the detection time of disease hotspots has decreased. Currently, osteoarthritis is not diagnosed at the level of radiographic changes in X-ray images - where they usually involved a large part of the joint and required treatment with total arthroplasty. Still, however, patients still cannot count on avoiding arthroplasty due to the lack of good tools for the treatment of small or medium articular cartilage defects. The next stage of treatment is therefore partial or superficial arthroplasty, which, unfortunately, also does not guarantee full recovery without the need to repeat the operation.

Complete endoprostheses still do not solve the problem of patient mobility, fitness and avoiding postoperative complications. The efficiency of the patient after the surgical procedure of total joint endoprosthesis implantation is significantly limited over the period of 3 months, the patients rarely return to full fitness. The level of patient satisfaction, measured by the degree of pain reduction and improvement in the function of the operated limb, varies. The main problems of post-operative arthroplasty are the level of pain, limb function, and acceptance of the effects of the operation itself. Excision of the joint means its final destruction - the next stage can only be an even more extensive excision of the tissues and implantation of an even larger implant - which in the second or third operation usually ends with a permanent disability.

For this reason, other therapeutic solutions are sought, especially among cell culture methods, in the field of transplantation of material obtained in cell culture, such as autologous chondrocyte transplantation.

"Premium" methods such as bearing technology and articular restoration have been introduced as a response to the need to ensure that patients can stay physically active as well as extend the time the implant must remain functional, but the cost of these technologies is much higher. The growing population of younger people who should be offered more expensive types of implants will lead to more and more burdens on health care systems.

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There is an increase in the number of endoprosthesis treatments all over the world. The sheer number of treatments is increasing, the patient population is affected by tissue degeneration leading to the need for these treatments, and the age at which the first treatment is performed is decreasing. The data presented below indicate the existence of a medical need to which the present invention pertains. The research conducted by Kurtz and colleagues (Kurtz SM et al. Future Young Patient Demand for Primary and Revision Joint Replacement: National Projections from 2010 to 2030, Clin Orthop Relat Res. 2009 Oct; 467 (10): 2606-2612) confirmed the thesis the increase in the population of younger patients. In 1993, 32% of primary and secondary total hip arthroplasty (THA) procedures were performed on patients under 65 years of age. In the case of knee implants, this value was approximately 26%. In 2006, there was an increase in the proportion of younger patients undergoing THA to 40-46% in terms of primary or secondary revisions. In the case of TKA (total knee arthroplasty, TKA), the values for primary procedures are 41%, and for secondary procedures - 44%. For patients under 65 years of age, clear increases in frequency are expected with primary THA as well as primary and secondary TKA. Currently, we are most likely aiming to exceed the barrier of 50% of procedures performed on younger patients. Calculations indicate that in 2030 primary THA performed in younger patients will constitute 52%, primary TKA 55%, secondary TKA 62%. The most intensive increase in the need for treatment is expected in the category of people aged 45-54, which is important from the social and economic point of view. In this group, the number of TKA (total knee arthroplasty) procedures in the US is expected to increase from approximately 59,000 in 2006 to approximately 994,000 in 2013, so an increase of 17 times is expected. The prognosis for THA (total hip arthroplasty) is a 5.9-fold increase in the number of operations. Projections for the number of knee prosthesis operations performed indicate a probable increase of 174% from 2005 to 2030, which will represent 572,000 procedures in the last year of the forecast. In the case of knee arthroplasty, the projected increase is expected to be even greater, amounting to 673% by 2030, which is approximately 3.5 million operations.

These increases necessitate the search for alternative treatments. These procedures are often long, straining the time of surgeons' work. In the event of such an intense increase in the demand for joint therapies, there is a risk of a significant extension of the waiting time for the procedure, in which, due to motor impairment and severe pain, patients will be unproductive, and will also put additional strain on social and health care systems.

One of the new methods is autologous chondrocyte implantation (ACI), which consists in taking a fragment of the patient's cartilage from the undamaged part of the joint, isolating the chondrocytes, multiplying them in vitro, and then implanting a suspension of cells in the place of cartilage damage.

The known methods of cell cultivation - chondrocytes for therapeutic purposes, consist in taking a piece of cartilage tissue, subjecting it to enzymatic digestion and then culturing the obtained cells - chondrocytes, with the use of a selected medium. Many enzymatic methods for obtaining a chondrocyte suspension for further cell culture are known. For this purpose, various types and combinations of enzymes are used, such as collagenase II, trypsin, and combinations of enzymes such as: collagenase II and hyaluronidase, trypsin and collagenase II, pronase and collagenase P. The chondrocytes obtained in this way are then transferred to the appropriate basic culture medium. . There are many culture media used for chondrocyte cultivation, such as: Dulbecco's modified Eagle's medium (DMEM) / Ham's F12- medium mixture, Dulbecco's modified Eagle's medium (DMEM) medium, Ham's F-12 medium or CMRL 1415 or RPMI 1640 medium. chondrocytes, supplements such as glutamine and additionally ascorbic acid, ITS-A (Insulin-Transferin-Selenium-A) or TGF-e1 and IGF-1 are also used.

Known culture media contain a number of components necessary for the proper growth of cells, such as: amino acids, salts, buffer components, glucose and additional components such as dyes. Depending on the medium, the concentrations and combinations of the listed components are different and specific.

DMEM (Dulbecco's Modified Eagles Medium) contains: calcium chloride anhydrous, iron nitrate 9ahydrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L -glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine

PL 240 714 B1 hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, calcium D-pantothenate, choline chloride, folic acid, i-inositol , niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride) with the sodium salt of 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid.

Ham's F-12 medium contains: calcium chloride anhydrous, cupric sulphate pentahydrate, ferrous sulphate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic anhydrous, zinc sulphate heptahydrate, L-alanine, L-arginine hydrochloride, L -asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride, L-glutamic acid, L-glutamine, glycine, L-histidine trihydrate monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium) , pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, linoleic acid, phenolic red, putrescine hydrochloride, pyruvic acid monobasic, thymidine.

The Trypsin-EDTA solution is used for passaging chondrocytes. It also happens that, apart from the basic medium, no supplements or antibiotics are added to the culture.

The selection of the basic culture medium and supplements and their use in appropriate concentrations or combinations is closely related to the desired goal. The scheme of arranging the culture medium looks different when focused on fast cell multiplication, differently when the goal is to inhibit chondrocyte differentiation or increase the production of type II collagen.

Chondrocyte culture can be adherent or suspension. Suspension is when the cells do not stick to the surface of the plate for the entire duration - they divide in suspension, the cells float in the medium.

It is known to cultivate chondrocytes in vitro adherent in a monolayer where cells are stuck to the surface of the plate. Both adherent and suspension cultures are mainly aimed at proliferating cells to the appropriate number and characteristics. The cells obtained in this way have a wide range of applications. From direct implantation in the form of a suspension to the patient from whom they were removed in the form of cartilage, to the formation of various types of structures that, only in the new form obtained, are transplanted into the patient.

There are known methods of obtaining cells for transplant in vitro, which involve the use of the so-called tissue glue. The following ingredients are used as the tissue adhesive: fibrinogen, thrombin, calcium chloride solution and aprotinin. The ingredients are applied with two syringes connected with a special connector. Within a dozen or so seconds from combining the components, an adhesive is formed, which, together with the chondrocyte suspension, forms a solution used for transplantation with adhesiveness at the site of the defect.

Cultures with the use of various types of compounds covering the surface of plates, bottles and other culture vessels in order to force the growth of cartilage cells on them seem to be more and more popular. The most common are: agarose, Poly-HEMA (poly-2-hydroxyethyl-methacrylate), alginate, Xano-Matrix nanofibers (made of polyethylene terephthalate and cellulose acetate), microspheres made of biodegradable PLGA (DL-lactic-co- glycolic acid) or natural and synthetic hydrogels. Adherent properties of chondrocytes are also used in conducting 3D culture with the use of a dedicated structure or skeleton, such as Gelfoam® (Pharmacia & Upjohn, Kalamazoo, MI) or BD ™ Three-Dimensional Collagen Composite Scaffold. The use of special microcarriers of various types enables a significant increase in the cultivation area and perfusion of the culture medium.

It is also known to use artificial structures that mimic naturally occurring bone and cartilage.

Known methods, however, do not allow for obtaining tissue with proper structure and biological features resembling natural tissue as much as possible.

Attempts to treat cartilage defects using biological therapy are the result of the methodology of cell culture of chondrocytes obtained from isolated tissue, and then administering them in suspension to the joint.

An example of this type of cell suspension product is Carticell. Treatment consisted of a cartilage biopsy during arthroscopy followed by cell expansion in vitro. After an appropriate time, the cells were returned to the joint under the periosteal flap during the second treatment. It was the first therapy of this type approved by the American FDA (Food and Drug Administration).

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The current generation of the product is called MACI (Genzyme Biotech). Clinical trials were conducted in a relatively small population. The endpoint was a minimum 10-point improvement in the KOOS Pain and Function (SRA) scales. In the MACI group, the response was 87.5% (63/72; 95% CI [77.6%, 94.1%]), while in the microfraction group it was 68.1% (49/72; 95% CI [56 0%, 78.6%]). There were fewer serious adverse events (13/72) in the MACI group than in the microfracture group (25/72).

A very similar product is ChondroCelect (TiGenix N.V., Belgium). The suspension for implantation is autologous human cartilage cells in DMEM containing amino acids, vitamins, salts, carbohydrates. The therapy also involves culturing cartilage cells ex vivo and then administering a suspension with a density of 10,000 cells / μl.

The biological treatment of ChondroCelect is supported by the level of tissue reconstruction compared to that achieved by the microfracture method measured 12 months after treatment. In addition, in a clinical trial, systematic improvement over 36 months was observed in the KOOS (the Knee Injury and Osteoarthritis Outcome Score) in both arms, although here the advantage of ChondroCelect was no longer marked. There was no statistical difference in the response of patients in the ChondroCelect arm and the control arm. The ChondroCelect (CC) group consisted of 41 patients, the group treated with the microfracture (MZ) method, 49 patients. Patients less than 3 years after the injury received a greater benefit in the CC group (27 out of 41) than in the MZ group (32 out of 49). After a period of 3 years, the differences between the groups had disappeared. Subsequent intervention in the joints treated in the clinical trial was required in 2 of 51 CC patients and 7 of 61 MZ patients. This was due to an insufficient degree of cartilage repair after treatment. After the study, the patients underwent long-term follow-up (5 years) - 37 in the CC arm and 40 in the MZ arm. In both arms, patients maintained the treatment effect noted at 36 months. There were no statistically significant differences between the groups. The cost of the therapy is approximately EUR 20,000 (https://bioinformant.com/price-of-cell-therapy-products).

The available methods of chondrocyte transplantation on collagen matrices are associated with a long period of treatment and convalescence. They are often burdened with a high failure rate above 50% - due to the fact that the damaged defect is not replaced with homogeneous tissue, but only partially with its components. The cost of the procedures, on the other hand, is very high. The therapeutic benefit / price ratio does not seem to be good for the patient.

Known methods of in vitro cultivation of chondrocytes for use in autologous transplants, i.e., multiplied cells in culture, and then administration of the suspension culture to the pond, do not guarantee the reconstructed joint, which is associated with a high cost and limited effectiveness compared to traditional surgical methods, difficulty in reconstructing tissue in vivo. Therefore, they are used especially in the treatment of minor injuries. The invention addresses the problems mentioned in the prior art.

Known chondrocyte cultures do not allow the proliferating cells to transform into cartilage. This is due to the difficulty of establishing intercellular connections - this requires time, ideal conditions for cell nutrition, and mechanical constraints.

As a result of experimental work, a methodology for cell culture of hyaline cartilage was developed in order to obtain material for the treatment of cartilage diseases. A method has been developed that involves the simultaneous use of native tissue fragments, which are joined in vitro into larger flaps, and a suspension of cells - chondrocytes, acting as an adhesive for joining tissue fragments.

Therefore, appropriately planned experimental studies were carried out to develop a methodology for carrying out chondrocyte cell culture, and, among other things, various cell densities, contact surface lengths of joined fragments, various types of supplements and culture media were tested to create intercellular junctions that are not easily disturbed.

According to the invention, cells grow and divide, stuck to the surface of the culture medium, e.g. plates. They are in suspension only when we detach them from the surface of the passage plate and when they are suspended in the medium, they are used to flood the tissue.

The invention relates to a methodology that allows the in vitro cultivation of cells and tissue fragments, so that it is possible to obtain cartilage tissue in a natural-like form rather than a suspension of cells or cells embedded in a matrix, as is known from the prior art. Cells and fragments of vitreous cartilage are cultured in vitro, and then the obtained tissue material is implanted in the patient's defect site. The invention is about

Taking smaller fragments of healthy cartilage from the patient from lightly loaded sites, and then placing them in a medium suitable for cell / tissue culture, where during cultivation the smaller fragments are combined into sheets of larger surface by direct insertion of at least two pieces of tissue - closely adjacent sections, i.e. so that the fragments touch at least on one edge of each of the sections, the tangent segment between the at least two tissue pieces to be joined being at least 0.1 cm. The parallel edges of the at least two tissue sections are shaped in such a way that the sections can be joined by joining together the mating edges on a tangential section where the fragments or entire edges of the edges are aligned in a parallel manner.

The method of carrying out in vitro cell culture of chondrocytes to obtain a material for the treatment of articular cartilage defects, is characterized according to the invention in that the collected vitreous cartilage is cut into at least two pieces of tissue, and then these fragments are placed on the culture plate next to each other in the culture medium. containing a minimum of 2 mM glutamine, minimum 2% FBS, minimum 4.5 g / l glucose, the shape of the tissue fragments is adjusted at least on one segment of the edge of each fragment so that, after placing the fragments close to each other, the space between at least two similar tissue fragments be as small as possible and so that the space of the culture plate filled with tissue fragments is as small as possible. The edges are adjusted in such a way that the fragments are joined together during the cultivation on at least one tangential segment. The tangent segment between the at least two tissue pieces to be joined is at least 0.1 cm. Chondrocyte suspensions are added to the culture and the chondrocyte tissue fragments are cultured in cells. The culture medium is changed at a predetermined time by adding chondrocytes to the culture medium, the culture being continued until the tissue fragment is joined at least part of the tangent segment. Preferably, the tangent segment between the at least two tissue pieces to be joined is at least 0.5 cm. Preferably, the tissue fragments are approximately oval in shape. Preferably, the shape of the tissue fragments is selected such that the tissue fragments in the culture adhere very closely to each other in the tangential segment.

Preferably, the culture medium contains supplements at a minimum concentration of 0.01% IGF and / or 0.01% TGF and / or 0.01% chondroitin and or / or a minimum of 0.1% insulin. Preferably, the culture medium comprises 5575 mg / L sodium pyruvate and sodium bicarbonate, 2 mM - 4 mM glutamine, 2% - 10% FBS, and at least 4.5 g / L glucose, as well as ethanolamine and / or glutathione and / or ascorbic acid and / or insulin and / or transferrin and / or copper sulfate and / or manganese chloride.

Preferably, prior to the addition of the chondrocytes, the tissue fragments themselves are cell cultured in medium, preferably for at least 14 days, after which the chondrocytes are added to the culture. Preferably, the chondrocyte suspensions are added to the culture of the tissue fragments in such an amount that there are at least 40,000 chondrocytes per 2.5 ml of the culture medium. Preferably, the minimum chondrocyte concentrations when culturing two fragments with a tangential section of 0.1 cm-1 cm are 40,000 chondrocytes per 2.5 ml of medium.

Preferably, the chondrocytes to be cultured are obtained by enzymatic digestion and the required number of cells is then obtained by culturing the chondrocytes in a monolayer in adherent culture.

Preferably, the cultivation is carried out under conditions of 37 ° C and 5% CO 2.

During the experimental work, it was experimentally established that the minimum tangent section at the joined edges must be at least 0.1 cm - a tangential section, i.e. the edges can be brought closer to each other on a parallel section. The tangent segment may be a portion of the edge of a tissue section or the entire edge of a tissue section. The edges to be joined together are in particular rectilinear or rounded. During cultivation, the sections are combined using a chondrocyte suspension culture obtained from the tissue of the same donor. The cartilage tissue contains both chondrocytes and the extracellular matrix. It is important to select the shape of the tissue fragments - sections so that the space between the edges of the joined pieces of cartilage is as small as possible - contact of the edge or edge section at least 0.1 cm. The matching of the shapes of the tangential edges between the sections - tissue fragments should take place at the latest at the stage of placing them in the immediate vicinity of the culture plate, before adding chondrocytes to them.

As a result, the culture enables the production of vitreous tissue and not only cells. At the site of joining cultured tissue sections, the resulting tissue may be less mechanically strong or less mature, but it will be a solid tissue with an intercellular junction.

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According to the invention, the procedure comprises:

1. Collection of vitreous tissue from the patient

2. Cutting the tissue into appropriate sections - fragments

3. Subjecting some of the sections to enzymatic digestion in order to obtain chondrocytes and further development of the cell culture - chondrocyte culture

4. Placing the remaining sections (non-digested) in the culture medium - culture of tissue fragments to which chondrocytes are added

5. After obtaining the appropriate number of chondrocytes in the culture, cells are obtained for further use in combined culture - fragments of tissue and cells, and the cells enable joining of non-digested tissue sections

6. Chondrocytes in suspension are added to the culture plate with the tissue fragments in it.

Suspension cells will locate in the fracture and help fuse at least two edges of at least two pieces of tissue.

In the combined culture, non-digested sections - tissue fragments in the culture are placed close to each other, matching the shape of the edges on the tangential section, so that the space between the joined edges is as small as possible, i.e. the tangential edges of the tissue fragments can be joined.

It is important to select the shape of the fragments - the lines of the tangential edges - so that the space between the edges of the adjacent pieces of cartilage is as small as possible. The matching of the shapes of the contact edges should take place at the latest at the stage of placing them in the immediate vicinity on the culture plate, before adding the chondrocytes to the suspension culture.

The invention was developed as a result of long-term experimental work. Examples 1-3 show trials of cell culture to obtain hyaline cartilage material for the reconstruction of damaged articular cartilage.

The invention is illustrated in the examples and in the drawing, in which Fig. 1 shows the principle of joining tissue fragments - A-B - tangential sections, C - shapes of tissue fragments, Fig. 2 shows the principle of joining tissue fragments - abnormal shapes of tissue fragments, in Fig. 3-5 - photos from the microscope.

Examples of connections that are not preferred are all types of arc-linear connections, as well as geometric connections, but with different angles, where the resulting space between the fragments has a relatively large volume, and there are no tangential segments running at least part of the edge of the scrap in a parallel manner on at least 0.1 cm is shown in Fig. 2. Fig. 2 shows examples of a non-preferred arrangement of the sections - the edges are not complementary over the entire course of the tangential sections, large volumes of space between the edges are created. Disadvantages: more difficult to fill the space, reduced mechanical resistance.

Example 1 - an attempt to cultivate only fragments of vitreous cartilage as carried out with the method according to the invention

In order to create a uniform flap of cartilage tissue as a material for the treatment of articular cartilage defects in the in vitro method, clinical material taken from an adult human was first obtained in the form of a fragment of hyaline cartilage taken from the knee joint, from unloaded sites. The tissue is collected from the patient from places with little movement load. This tissue should be healthy. The shape of the removed cartilage flap or flaps may therefore vary. One or more pieces of cartilage may be harvested. One or more fragments can be subdivided into smaller sections as slices across or along the cartilage-bone line.

Next, the cartilage fragment was cut into smaller pieces under sterile conditions in the case of collecting a large piece of articular cartilage.

Immediately after taking the material from the patient, it was transferred to a sterile 50 ml tube filled with saline solution (0.90% NaCl) so that the tissue was completely immersed in the solution. Then the cartilage was thinly cut to a thickness of max. 3 mm slices of various shapes and sizes less than 1 cm in length and / or width or thickness.

The obtained tissue fragments, completely immersed in saline solution (0.90% NaCl), were transported in a sterile 50 ml tube to the cell culture laboratory. Tissue transport was performed under sterile conditions at 2-8 ° C. All tissue operations were performed under laminar flow conditions. After preparing patches of an appropriate size, the obtained material was the starting point for the final tissue flap.

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The final tissue flap for the treatment of articular cartilage defects was to be formed on a matrix of tissue slices that were properly aligned on the surface of the well of the culture plate, grown under appropriate conditions and the composition of the culture medium.

An important step was to properly adjust the shape of the edges of the tangential slices of tissue. By joining the tissue fragments with tangential edges, the tissue fragments were arranged in the closest relation to each other on the culture plate.

A cultivation trial was carried out with the use of tissue fragments from 2 to 4 touching each other on at least one rectilinear section - the tangent edge from a length of 0.1-1 cm.

As previously described, the shape was fitted according to the tangent edges of at least two tissue fragments - a section - of the joined edges. The maximum length / width and the maximum number of fragments - a section depends on the size of the culture plate.

Of all the slices of the harvested tissue material, it was necessary to select a minimum of two slices up to the maximum number that would fit on the well surface of the culture plate used. In the event that the above-mentioned arrangement of prepared patches is not possible due to the mismatch of the edges of the patches so that they adjoin each other, they are trimmed and thus given the desired shapes after the material is taken from the patient.

It was assumed that at these stages of tissue culture, where it is planned to start creating a new tissue fragment based on previously collected and prepared tissue fragments, it is crucial to arrange at least two tissue fragments next to each other. These fragments should be arranged so that the joined fragments have tangent edges over the longest possible segment and are compatible in shape with the adjacent fragment (s), so that the filling space of the culture plate is as small as possible. This effect can be obtained both at the stage of collecting tissue from the patient by collecting fragments of an appropriate shape, and later in the laboratory in in vitro conditions. Edge shape compatibility - their location in the vicinity so that at least one edge of one tissue fragment touches the edge of the other tissue, which ensures easier filling of the surface between them by suspended chondrocytes thanks to minimizing the volume of free space between cartilage fragments, as well as increasing resistance mechanical damage of the newly obtained tissue fragment at the connection site.

During cultivation, it was observed whether the applied tissue flaps - sections - would increase the surface area and whether the spaces between the edges would be filled. Compatible tissue slices approximately 1 mm x 1.2 cm x 0.7 cm were transferred to the wells of a 6-well sterile cell culture plate filled with 2.5 ml of cell culture medium 2 containing: calcium chloride anhydrous, sulfate cupric pentahydrate, ferrous sulfate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic anhydrous, zinc sulfate heptahydrate, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid L-cysteine hydrochloride , L-glutamic acid, L-glutamine, glycine, L-Histidine trihydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine , L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, linoleic acid y, phenol red, putrescine hydrochloride, monobasic pyruvic acid, thymidine with the addition of a 1-fold concentrated solution Glutamax supplement. The cultivation was carried out under the conditions of 37 ° C and 5% CO 2 for 7 days. Links between the two tissue fragments, viability and proliferation were monitored with 10x and 40x optical magnification. Tissue modifications: fusion, viability and proliferation were observed regularly, prior to each subsequent passage and replacement of the medium with fresh media, using an inverted optical microscope at 10x and 40x magnification. The culture medium and supplements were changed weekly.

With each subsequent change of the medium, the following occurred:

1) removal of the old medium

2) gently transferring the tissue slices to a new culture plate

3) addition of fresh medium.

During cultivation, it was observed whether the applied tissue flaps - sections - would increase the surface area and whether the spaces between the edges would be filled. After five months of culturing the tissue fragments only - without the chondrocyte suspension, by proceeding as in the invention, no connections were observed between the pairs of cartilage fragments placed in one well. Nor was there any new tissue growing on top of the old tissue.

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Example 2 - an attempt to cultivate only fragments of cartilage carried out by the method according to the invention

The culture procedure was similar to that described in Example 1.

The compatible tissue slices were transferred to the wells of a 6-well sterile cell culture plate filled with 2.5 ml of cell culture medium 2 supplemented with a 1-fold concentrated Glutamax Supplement solution and glucose [4.5 g / L].

After five months of cultivation, no connections were observed between the pairs of pieces of cartilage placed in one well. Nor was there any new tissue growing on top of the old tissue.

Example 3 - an attempt to cultivate only fragments of cartilage carried out by the method according to the invention

The culture procedure was carried out as described in Example 1 except that compatible tissue slices were transferred to the wells of a 6-well sterile cell culture plate filled with 2.5 ml of cell culture medium containing: calcium chloride anhydrous, iron nitrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine , L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, calcium D-pantothenate, choline chloride, folic acid, i-inositol , niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride with 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt with high glucose concentration [4.5 g / l].

After five months of cultivation, no connections were observed between the pairs of pieces of cartilage placed in one well. Nor was there any new tissue growing on top of the old tissue.

Example 4 - General Description of the Process of the Invention

In order to create a uniform flap of cartilage tissue as a material for the treatment of articular cartilage defects in the in vitro method, clinical material taken from an adult human was first obtained in the form of a fragment of hyaline cartilage from the knee joint. The hyaline cartilage is always collected preferentially from the treated joint (it can be knee, hip, ankle, shoulder, any other joint). It can be collected from another joint in a situation where the treated joint is small or it is difficult to collect hyaline cartilage from it. It should always be an unencumbered place. The tissue is collected from the patient from places with little movement load. This tissue should be healthy. The shape of the removed cartilage flap or flaps may therefore vary. One or more pieces of cartilage may be harvested. One or more fragments can be subdivided into smaller sections as slices across or along the cartilage-bone line.

Next, the cartilage fragment was cut into smaller pieces under sterile conditions in the case of collecting a large piece of articular cartilage.

Immediately after taking the material from the patient, it was transferred to a sterile 50 ml tube filled with saline solution (0.90% NaCl) so that the tissue was completely immersed in the solution. Then the cartilage was thinly cut to a thickness of max. 3 mm slices of various shapes and sizes less than 1 cm in length / or width (this applies both to the fragments that will be the basis for obtaining the final tissue sheet and the fragments that will give rise to free chondrocyte culture.

Possible sizes are from about thickness / length / width: 1 mm x 0.5 cm x 0.5 cm, 1 mm x 0.6 cm x 0.6 cm, 1 mm x 0.7 cm 0.7 cm, 1 mm x 0.8 cm x 0.8 cm, 1 mm x 0.9 cm x 0.9 cm, 1 mm x 1 cm x 1 cm, 2 mm x 1 cm x 1 cm, 3 mm x 1 cm x 1 cm. The obtained tissue fragments, approx. 1 mm x 1 cm x 1 cm in size, completely immersed in saline solution (0.90% NaCl), were transported in a sterile 50 ml tube to the cell culture laboratory. Tissue transport was performed under sterile conditions at 2-8 ° C.

The dimensions and shape of the panels to be joined do not matter. It is important to prepare them so that the space between the joined edges on the tangential section of at least two pieces of tissue is as small as possible, so that the edges are joined together by at least 0.1 cm of the length of the tangential section. The size of the fragment itself also depends on the size and shape of the injury as well as on the size and shape of the healthy cartilage removed to obtain a "biological prosthesis" in the form of a reconstituted piece of cartilage according to the invention. The tangent segment was at least 0.1 cm. All

The operations are performed with tissue or cells performed under laminar flow conditions. After preparing slices of the appropriate size from them, all fragments were divided into two parts. One of them was used to obtain a chondrocyte suspension, i.e. a mixture that acts as a tissue building material in the culture of cells with tissue fragments. The second was the fragments for the cultivation of tissue flaps and was the basis for obtaining the final flap of reconstituted tissue. The final tissue flap for the treatment of articular cartilage defects was obtained according to the procedure described below, that is, tissue slices of a fixed shape, suitably positioned against each other on the surface of the well of the culture plate, were combined by means of a chondrocyte suspension - in a "tissue cell" culture.

1) Preparation of pieces of cartilage tissue for further cultivation of tissue pieces.

An important step is the proper selection of those tissue slices, the edges of which will allow them to be placed as closely as possible to each other on the culture plate. Tissue fragments must touch each other over at least one rectilinear segment. The minimum number of tissue fragments in a section is two. The minimum length for a piece of tissue of any shape: minimum: 0.1 cm, preferably minimum 0.5 cm. The minimum width for a piece of tissue of any shape: minimum: 0.1 cm, preferably minimum 0.5 cm. Tissue thickness: minimum 1 mm and maximum 4 mm.

As previously described, the shape follows the tangential edges of at least two tissue fragments - sections - of the joined edges on the tangential segment. This length of the tangent section of the joined edges should be at least 0.1 cm, preferably at least 0.5 cm, regardless of whether the section is straight or curved, since this length relates to the tangent line of the tangential section and not a straight line. The maximum length / width as well as the maximum number of fragments - sections depends on the size of the culture plate and the therapeutic target.

Of all the slices of the harvested tissue material, it was necessary to select a minimum of two slices up to the maximum number that would fit on the well surface of the culture plate used. In the event that the said arrangement of prepared patches is not possible due to the mismatch of the edges of the patches so that they adjoin each other, it is possible to trim them and thus give the desired shapes after the material is taken from the patient.

At these stages of tissue and cell culture, where it is planned to start creating a new tissue fragment based on previously collected and prepared tissue fragments, it is crucial to arrange at least two tissue fragments next to each other. These fragments should be arranged so that the joined fragments have tangential edges over the longest possible segment, but at least 0.1 cm, compatible in terms of shape with the adjacent fragment (s), so as to minimize the volume of free space between the cartilage fragments and so that the plate space is filled. livestock was as small as possible. This effect can be obtained both at the stage of collecting tissue from the patient by collecting fragments of an appropriate shape, and later in the laboratory in in vitro conditions. Edge shape compatibility - their location in the vicinity so that at least one edge or its fragment of one tissue fragment touches the edge of the other tissue fragment, which ensures easier filling of the surface between them by suspended chondrocytes thanks to minimizing the volume of free space between the fragments of cartilage, as well as increases the mechanical resistance of the newly obtained tissue fragment at the connection site. Possible examples of edge shapes and methods of joining them are shown in Fig. 1, the examples are not exhaustive of the list of all possibilities. It is important to obtain and combine during cultivation a minimum of two pieces of tissue directly adjacent to each other with edges on the adjacent edge section, where the minimum tangent section is 0.1 cm for both straight and curved sections. This value relates to the length of the tangent segment of the fragment or the entire edge of the tissue fragment, regardless of its shape.

Fig. 1 shows the entire fragments of tissues or the edges of the fragments of tissues - sections, showing their connection by bringing together the matching edges on the tangential section.

According to the invention, it is important to adjust the shape of a piece of tissue - a piece of tissue by planning the course of adjacent edges in parallel - the place where at least two adjacent tissue fragments are joined. This shape exists for all shapes with straight sides. This shape may also be straight and then arcuate over a certain portion. The shape of the tissue fragments themselves - the scraps, therefore, can be any. The course of the edge line should be compatible on both joined tissue fragments in the tangential section - parallel so that both tissue fragments can be joined at these edges. Edge section or all

The edge of the joined tissue fragments on the tangential section - in the section where at least two pieces of tissue connect, is hereinafter referred to as the tangential section.

The rule is that the tangential length of the joined edges of the tissue - the length of a part or the entire edge of the fragment on the tangential section - the joining of tissue fragments should be at least 0.1 cm of the edge - the length of the tangential segment. Fig. 1 shows exemplary shapes of the tangent sections: A - rectilinear sections, B - arched sections, as well as examples of shapes of the joined tissue fragments: C - angular and geometric tissue fragments, their combinations.

Examples of connections that are not preferred are all types of arc-linear connections, as well as geometric ones, but with different angles, where the space created between the fragments has a relatively large volume and there are no tangential segments running at least a fragment of the edge of the scrap in a parallel manner along the segment. at least 0.1 cm is shown in Fig. 2. Fig. 2 shows examples of a non-preferred arrangement of the scraps - the edges are not complementary over the entire course of the tangential sections, large volumes of space between the edges are created. Disadvantages: more difficult to fill the space, reduced mechanical resistance.

2) Initial culture of the tissue fragments only.

Selected or appropriately cut slices - tissue fragments - sections, as described previously, were transferred with forceps to the wells of a 1-well, 6-well or 12-well sterile cell culture plate. Selected or appropriately cut tissue slices were placed in the wells previously filled with 10 ml, 2.5 ml or 1.25 ml of cell culture medium 1 with 4.5 g / l glucose also called DMEM or medium 2 with the addition of the solution 1-fold concentrated solution Glutamax supplement called the trade name F12 or a mixture of medium 1 and medium 2 in the ratio of 1: 1 and 2%, 4%, 5% or 10% FBS serum and 2 mM - 4 mM glutamine.

For this example, culture medium 1, known under the trade name DMEM, was used, containing: calcium chloride anhydrous, iron nitrate 9ahydrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride , L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan , disodium L-tyrosine dihydrate, L-valine, calcium D-pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride with sodium salt of 4- (2-hydroxyethyl) piperazine-1- ethanesulfonate with the addition of glucose.

For this example, the culture medium 2 was used, containing: calcium chloride anhydrous, cupric sulfate pentahydrate, ferrous sulfate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic anhydrous, zinc sulfate heptahydrate, L-alanine, L-arginine hydrochloride , L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride, L-glutamic acid, L-glutamine, glycine, L-Histidine trihydrate monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium ), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, linoleic acid, phenolic red, putrescine hydrochloride, monobasic pyruvic acid, thymidine with the addition of a 1-fold concentrated solution of Glutam supelmet. x.

For this example, the performance of the 1: 1 mixture of both media was also checked.

It is essential that the medium contains a minimum of 4.0 g / L glucose, preferably 4.5 g / L glucose, a minimum of 10% FBS, a minimum of 2 mM glutamine.

It is preferred that the culture medium contains 55-75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM - 4 mM glutamine, 2% - 10% FBS, and at least 4.5 g / L glucose, as well as ethanolamine, glutathione, acid. ascorbic, insulin, transferrin, copper sulfate, manganese chloride.

In each well of the culture plate used, a minimum of two slices of tissue, approx. 1 mm x 1 cm x 1 cm, were placed so that one of the edges adhered maximally on the tangential edge section or the edge fragments on the tangential section in the example made at least 0.1 cm. Several experimental experiments were carried out with the use of 2-4 pieces with a tangent section of 0.1-0.5-1 cm. The cartilage was incubated for 7 days at 37 ° C and 5% CO 2. After this time, microscopic observations were made and the medium was replaced with fresh ones.

PL 240 714 B1

For the 6-well plate used in this experiment, the volume of fresh medium each 7 days was changed to 2.5 ml. An inverted microscope was used to evaluate the viability of the tissue patches; microbiological purity of the culture; control of the appearance of possible new cell structures resulting from the division of cells present in the tissue. Links between the two tissue fragments, viability and proliferation were monitored with 10x and 40x optical magnification.

The tissue fragments alone were cultivated until a sufficient number of chondrocytes was obtained (separate culture - adherent culture) to flood the tissue fragments with the suspension of multiplied chondrocytes. For this experiment, it was 17 days. The cultivation of the tissue fragments themselves was a kind of incubation and was aimed at keeping the tissue fragments active and ready for the next step already in proper culture with free chondrocytes.

3) Obtaining a suspension of chondrocytes and initial adherent culture of the chondrocytes themselves.

The production of chondrocytes and the cultivation of chondrocytes for their multiplication can be carried out by a known method.

After selecting the tissue patches as the basis of the future new tissue flap, the remaining patches for the isolation of chondrocytes, constituting from 15% to 50% of the weight of all patches, were used to obtain the chondrocyte suspension. The shape of the piece of tissue for obtaining chondrocytes does not matter. For this purpose, the tissue intended for enzymatic digestion was additionally shredded into smaller pieces (not larger than 3 mm x 3 mm x 3 mm) using a scalpel. The material prepared in this way was treated with collagenase II.

The resulting pieces of tissue were transferred to a sterile 15 ml to 50 ml tube. In this case it was a 50 ml tube. Tissue pieces were rinsed 3 times with 1X concentrated phosphate buffered saline (1 x PBS). The time of enzymatic digestion, as well as the amount of enzyme, was adjusted to the weight of the tissue fragments as well as to obtain proper cell viability after the reaction. The following study used tissue to enzyme weight ratios ranging from: 1 g: 1 mg, 1 g: 2 mg, 1 g: 3 mg and 1 g: 4 mg. The digestion reaction was carried out under the conditions of culture medium 1, known under the trade name DMEM, containing: calcium chloride anhydrous, iron nitrate nine-hydrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L- tryptophan, disodium L-tyrosine dihydrate, L-valine, calcium D-pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride with sodium salt of 4- (2-hydroxyethyl) piperazine-1 -ethanesulfonic acid with the addition of glucose;

or under the conditions of the culture medium 2 containing: calcium chloride anhydrous, cupric sulfate pentahydrate, ferrous sulfate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic anhydrous, zinc sulfate heptahydrate, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride, L-glutamic acid, L-glutamine, glycine, L-Histidine trihydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium ), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, linoleic acid, phenolic red, putrescine hydrochloride, pyruvic acid monobasic, thymidine with the addition of a 1-fold concentrated solution of Glutamax supelmet;

or a mixture of the two above-mentioned growth media used in a ratio of 1: 1 with the addition of glucose.

The tissue was placed in the medium in the ratio: 3 ml of medium: 0.1 g of tissue or 3 ml of medium: 0.2 g of tissue or 3 ml of medium: 0.3 g of tissue or 3 ml of medium: 0.5 g of tissue, containing the appropriate concentration collagenase II. Tissue weight in grams is the sum total of the weight of the tissue patches used. The maximum digestion was performed on the slices, the total weight of which was 0.5 g. Digestion was performed under the conditions of 37 ° C, 5% CO2 in a cell culture incubator. The reaction was carried out for a minimum of 16 hours until the tissue was completely digested. After digestion, the supernatant containing free chondrocytes was removed and centrifuged at 300 x g for a minimum of 10 minutes at room temperature. The obtained cell pellet, containing chondrocytes in the amount of 0.3 million from 1 g

Tissues up to 1.5 million from 1 g of tissue were washed three times in 1 fold concentrated PBS. The amount of tissue subjected to digestion is usually approx. 0.1 - 0.2 g on average. Finally, the cell pellet was resuspended in at least 1 ml of medium 1 with 4.5 g / l glucose or medium 2 with the addition of a solution once concentrated Glutamax supplement or a mixture of medium 1 and medium 2 in the ratio 1: 1, containing 2% or 4% or 5% or 10% FBS and a minimum of 2 mM glutamine solution for the purpose of observation and qualitative and quantitative evaluation of the digestive reaction. It was assessed whether the cells were viable after digestion (qualitative assessment) and whether the entire tissue fragment had been digested (quantitative). The cell viability was qualitatively assessed (at least 80% viability was considered positive), the shape and general condition of the cells after digestion (round cells with a visible nucleus were considered in good condition). Cells with a viability of not less than 80% were seeded at densities of 150,000 cells per 2 ml of medium or 200,000 cells per 2 ml of medium or 250,000 cells per 2 ml of medium in 25 cm ² or 75 cm ² culture bottles. During cultivation, the cells were observed every other day with an inverted optical microscope to assess the growth rate and determine the time of cell passage closely related to the level of confluence. The culture medium was changed twice a week. When the cells were sufficiently confluent (85% maximum), they were trypsinized for 5 minutes with 2 ml of a 0.05% (w / v) trypsin / EDTA solution in PBS at 37 ° C. Inhibition of trypsinization was carried out by adding medium 1 with the addition of glucose 4.5 g / l or medium 2 with the addition of a 1-fold concentrated solution of Glutamax Supplement or a mixture of medium 1 and medium 2 in the ratio 1: 1, containing 2%, 4%, 5 % or 10% of FBS in a volume of 1, 1.5 or 2 times the volume of the trypsin / EDTA solution used. Cell number and viability were determined with a microscope and trypan blue solution according to standard procedure.

During the cultivation of chondrocytes, first of all, cell growth and viability were monitored. The cultivation was carried out until reaching the confluence of 85% with min. 80% of cell viability. This culture was carried out in order to multiply a sufficient number of cells for the next culture of the joined tissue fragments, including the chondrocytes themselves, i.e. the so-called "Tissue potting", where the chondrocyte suspension serves as an adhesive to connect the tissue fragments used. Some of the cells were used to "flood" the tissue and some to establish another culture so that cell material was still available.

Cells were kept in continuous culture to be used to stimulate the connections between pieces of cartilage described in a).

The cultivation of chondrocytes in order to multiply them is always carried out in a monolayer - adherent culture. We refer to a suspension of cells when, after detaching the cells from the surface of the plate, the described steps of washing and centrifuging are performed, and the chondrocytes are suspended in the target buffer and flooded with them in the tissue flaps in the combined culture.

4) Combined culture - fragments of tissue with a suspension of chondrocytes obtained cells according to point 3

It is important to add a minimum of 40,000 chondrocytes to a well containing 2.5 ml of the culture medium, assuming the use of two slices of the tank of approx. 1 mm x 1 cm x 1 cm. The maximum concentration of chondrocytes when culturing two fragments with a tangential section of 0.1 cm is 8,000 cells per 2.5 ml of medium.

Cartilage tissue slices, cultured in medium 1 with 4.5 g / l glucose or medium 2 with the addition of a 1-fold concentrated solution of Glutamax supplement or a mixture of medium 1 and medium 2, described above, in a ratio of 1: 1 to 2% or 4 % or 5% or 10% FBS, with a minimum of 2 mM glutamine and a minimum of 0.01% IGF-1 and / or a minimum of 0.01% TGF-e1 and / or a minimum of 0.01% chondroitin and / or a minimum of 0.1 % of insulin were "flooded" with chondrocytes, obtained by their cultivation after digestion (viability> minimum 85%).

It is essential that the medium contains a minimum of 4.5 g / L glucose, a minimum of 10% FBS, and a minimum of 2 mM glutamine.

It is preferred that the culture medium contains 55 - 75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM - 4 mM glutamine, 2% - 10% FBS, and at least 4.5 g / L glucose, as well as ethanolamine, glutathione, acid. ascorbic, insulin, transferrin, copper sulfate, manganese chloride and a minimum of 0.01% IGF-1 and / or a minimum of 0.01% TGF-e1 and / or a minimum of 0.01% chondroitin and / or a minimum of 0.1% insulin.

After centrifugation and prior quantitative and qualitative evaluation performed as described above, the cells were suspended in medium 1 with 4.5 g / l glucose or medium 2 with 1 times the solution.

Using a concentrated Glutamax Supplement solution or a mixture of medium 1 and medium 2 in a 1: 1 ratio in a minimum volume of 500 μl for a minimum of 40,000 cells, then added to a well with a minimum of two pieces of tissue. It is important that the culture medium contains 55 mg / l - 75 mg / l sodium pyruvate, sodium bicarbonate, 2 mM - 4 mM glutamine, 2% - 10% FBS, and 4.5 g / l - 5.0 g / l glucose. .

The resulting combination of tissue fragment and chondrocyte culture was run under the conditions of 37 ° C, 5% CO 2 in a cell culture incubator for a minimum of 12 weeks until new tissue connections were obtained between the tissue fragments used. The process of "flooding" the tissue with the suspension, each time containing a constant amount of chondrocytes, was repeated once a week. The exchange of the medium with the indicated frequency was dictated by the experience gained in conducting tissue cultures. The addition of chondrocytes took place after the then gentle transfer of tissue fragments into a new well of a 6-well plate. At that time, the medium was also replaced with fresh medium, in the case of a 6-well plate it was the final volume with supplements equal to 2.5 ml.

Tissue modifications: fusion, viability and proliferation were observed regularly, before each successive "flooding" of tissue with cells, using an inverted optical microscope at 10x and 40x magnification. The culture medium and supplements were changed weekly.

With each subsequent "flooding" with new chondrocytes, the following occurred:

1) Removal of the old medium

2) Gently transfer the tissue slices to a new culture plate

3) Addition of fresh medium

4) Pouring the slices with cells (as above given the number of cells and the medium in which they are suspended - now the volume of the fresh medium has been added) - description above

After a few days of test culture, visible changes in the cartilage tissue were noticed. New tissue connections between old tissue fragments were identified. After a few weeks, the gap between the old tissue fragments was filled with new tissue and overgrown. A new tissue flap was obtained, made of cartilage slices and free chondrocytes regularly supplied to the culture. The resultant one piece of tissue from the original two slices of tissue had a "groove" in the center, which was supposed to be new tissue, formed by chondrocytes regularly supplied to the culture as a suspension. The tissue flap was approximately 1 mm x 2.1 cm x 1 cm. It was possible to transfer it from the well of the plate to another destination without the need to use two tweezers and without the risk of damage. The new connection between the original tissue slices was slightly lighter in color than the old tissue as shown in Figure 3 - inverted microscope photo on the day of starting the culture (Figure 3A), after seventeen (Figure 3B) and fifty-two days (Figure 3B). Figs. 3C, D) cell-tissue culture using two pieces of tissue with a tangential section.

Throughout the period of proper cultivation, which was a combination of tissue slice culture and cell suspension, the chondrocyte suspension was cultivated separately in parallel as described above. Culturing the cells in suspension was necessary to continuously maintain the source of cell material used to "pour" the tissue slices. The observations were made at a magnification of 40 times.

Fig. 3 shows a microscopic image of the culture of the tissue slices in medium 1 with the addition of 4.5 g / L glucose, 10% FBS, 2 mM glutamine as described above - visualization of new tissue formation over time. A: Image of fragments of two tissue slices adjacent to each other as closely as possible on the tangential section of approx. 1 cm in length on the first day of culture (photo documenting the start of culture, day 1). Clearly visible free space between the contact surfaces of the two tissue patches. B: Image of fragments of two tissue patches, adjacent to each other on a tangential section of approx. 1 cm and visible tissue filling, formed between the contact surfaces of two tissue patches on the seventeenth day of culture in medium 1 with the addition of 4.5 g / l glucose, 10% FBS, 2mM glutamine. C, D: Image of fragments of two tissue patches, adjacent to each other on a tangential section of approx. 1 cm and a visible tissue filling, formed between the contact surfaces of two tissue patches on the fifty-second day of culture in medium 1 with the addition of 4.5 g / l glucose, 10% FBS, 2 mM glutamine. The new tissue formed after fifty-two days of cultivation clearly scarifies the free space present at the junction of two tissue slices on the first day of cultivation. Visible significant alignment of the color of old and new tissue on one of the ends of the two contact surfaces of the visible fragments of tissue patches. Pictures taken with the use of a 10x magnification (A, B, C) and a 40x magnification (D).

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Example 5 - an embodiment of the invention

The cultivation is carried out as described in Example 4, except that the conditions are changed to a certain extent.

1) Obtaining pieces of cartilage for further cultivation

Eight slices of tissue with an almost oval shape were obtained, but on one side each panel was trimmed to allow the greatest possible contact area for the two pieces placed next to each other. The obtained slices were thin, approximately 1 mm thick. They are 0.9 cm long and 0.5 - 0.6 cm wide.

2) Initial culture of the tissue fragments only

Selected and appropriately cut slices as previously described were transferred with forceps to the wells of a 6-well sterile cell culture plate. Two selected slices of tissue, approx. 1 mm thick and approx. 0.9 cm long and approx. 0.6 cm wide, were placed in a well previously filled with 2.5 ml of the mixture of medium 1 and medium 2 with the addition of 4.5 g / l of glucose and 10% serum FBS, 55 mg / L sodium pyruvate and 2 mM glutamine. The tangent was rectilinear. Medium 1 and 2 are described in the previous example.

In the three wells of the used culture plate, two slices of tissue were placed in each, arranged so that one of the edges (tangent edge) approx. 0.6 cm long adjoined each other. They were biological replicates of the same test. The cartilage was incubated for 7 days at 37 ° C and 5% CO 2. The culture medium was changed every 7 days, each time using an inverted microscope to evaluate the viability of the tissue patches; microbiological purity of the culture and analysis of the appearance of any new cell structures resulting from the division of cells present in the tissue. 10-fold and 40-fold optical magnification were used. Observations were made before the medium was changed.

3) Obtaining a chondrocyte suspension and culturing

The other two patches (size: approx. 1 mm x 0.9 cm x 0.5 cm) were intended for the isolation of chondrocytes, they constituted a total of approx. 25% of the weight of all patches. For this purpose, the tissue intended for enzymatic digestion was additionally shredded into smaller pieces (the size of the pieces obtained approx. 2 mm x 2 mm x 2 mm) using a scalpel. The material prepared in this way was treated with collagenase II. The resulting pieces of tissue were transferred to a sterile 30 ml tube. Tissue pieces were rinsed 3 times with 1X concentrated phosphate buffered saline (1 x PBS). The following study used tissue to enzyme weight ratios: 1 g: 1 mg. The digestion reaction was carried out under the conditions of a mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose (in the ratio of 3 ml of medium 0.3 g of tissue - i.e. 2 ml), containing the appropriate amount of collagenase II, i.e. 0.2 mg. Tissue weight in grams was the total value of the weight of the tissue slices used. Digestion was performed under the conditions of 37 ° C, 5% CO 2 in a cell culture incubator. The reaction was run for 20 hours overnight. After digestion, the supernatant containing free chondrocytes was removed and centrifuged at 300 xg for 10 minutes at room temperature (21 ° C). The obtained cell pellet, containing chondrocytes, was washed three times in 1-fold concentrated PBS. Finally, the cell pellet was resuspended in 1 ml of the mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose, containing 10% FBS and 2 mM glutamine for the purpose of observation and qualitative evaluation (mainly evaluation of the viability and condition of cells after digestion - e.g. regularity of shape) and quantitative digestion reaction. A 98% viability of chondrocyte suspension was obtained, containing 0.49 million viable cells. The obtained cells were then seeded at a density of 10,000 cells per cm ² . Cells were seeded in 6-well plates and in a 25 cm ² culture flask.

Cells were kept in continuous culture for use in stimulating connections between pieces of cartilage.

4) Combined culture - fragments of tissue with a suspension of chondrocytes

The cartilage tissue patches described in item 2 of this example, grown in the mixture of medium 1 and medium 2 with the addition of 4.5 g / L glucose, 10% FBS, 2 mM glutamine, 55 mg / L pyruvate were "flooded" with chondrocytes obtained as a result of their cultivation after digestion (viability in the range of 85% - 92%). The cells, after centrifugation and prior quantitative and qualitative evaluation performed as described above (point 3 of this example), were suspended in a mixture of medium 1 and medium 2 with 4.5 g / l of glucose in a final volume of 1000 μl, followed by a volume of suspension containing 80 000 cells were added to each well with two tissue slices. Supplements were then added which in this study were IGF-1 and TGF at a final concentration in the medium in the well for both additives of 0.01%.

PL 240 714 B1

The resulting tissue fragment and chondrocyte culture combination was incubated at 37 ° C, 5% CO 2 in a cell culture incubator for a total of 5 weeks until new tissue connections were established between the tissue fragments used. The process of "pouring" the tissue with a suspension containing each time a constant amount of chondrocytes (80,000 / 2.5 ml of cell medium per well, containing two tissue patches) was repeated once a week (every 7 days). Before the tissue was transferred and fresh medium and chondrocytes were added, the tissue slices were observed (as described in Example 4). The addition of chondrocytes took place after the then gentle transfer of tissue fragments into a new well of a 6-well plate.

After seven days of test culture, the first visible changes in the cartilage tissue were noticed. After nineteen days, new tissue junctions were identified between the old tissue fragments as shown in Fig. 4 - inverted microscope photo on the day of starting the culture (Fig. 4A) and after nineteen days of cell-tissue culture (Fig. 4B) using two pieces of tissue with a tangential section of 0.6 cm. The new connections resembled the fibers that were formed between the patches of tissue, binding the patches. After five weeks, the gap between the old pieces of tissue was filled with new tissue and overgrown. The junction of the two tissue patches was filled with new cell connections. The new tissue between the cartilage patches took the form of a kind of "scar". On microscopic images, the new tissue is brighter than the tissue of the original cartilage patches. A new tissue flap was obtained, made of cartilage slices and free chondrocytes regularly supplied to the culture. A new tissue flap was obtained, approximately 1 mm x 1.8 cm x 0.6 cm in size.

Fig. 4 shows a microscopic image of the culture of the tissue slices in the mixture of medium 1 and 2 - visualization of the formation of new tissue connections over time. A: Image of fragments of two tissue slices adjacent to each other as closely as possible, over a distance of 0.6 cm on the first day of culture (photo documenting the start of culture). Clearly visible free space between the contact surfaces of the two tissue patches. B: Image of fragments of two tissue patches, adjacent to each other over a distance of 0.6 cm and visible tissue connections formed between the contact surfaces of two tissue patches on the nineteenth day of culture in the mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose, 10% FBS, 2 mM glutamine and supplements: IGF-I and TGF at a concentration of 0.01%. Visible new tissue, both on the surface of one of the adjacent pieces and between the pieces of tissue patches. The resulting connections are fibers connecting the two contact surfaces with each other. Photo taken using a 10x zoom.

Example 6 - an embodiment of the invention

Cultivation is performed as described in Examples 4 and 5, except that:

1) Obtaining pieces of cartilage for further cultivation:

Ten slices of tissue approximately oval in shape were obtained, except that each flap was trimmed on two sides (the two longest). The obtained slices were about 3 mm thick. They are about 1 cm long and 0.2 cm wide.

2) Initial culture of the tissue fragments only:

Selected and appropriately cut slices as previously described were transferred with forceps to the wells of a 6-well sterile cell culture plate. Four selected slices of tissue, approx. 3 mm thick and approx. 1 cm long (length of the contact surface - in each fragment the shape of the surface ensured the maximum surface area and a compatible contact surface, similar to a rectilinear one) and a width of approx. 0.2 cm were placed in the well previously filled 1.25 ml of the mixture of cell culture medium 1 and 2 with the addition of 4.5 g / L glucose, 2% FBS serum and 2 mM glutamine.

The tangent segment between each pair was approx. 1 cm. In a well of the 12-well culture plate used, four slices of tissue were placed, arranged so that one of the edges (tangent edge) approx. 1 cm long adjoined each other.

3) Obtaining a chondrocyte suspension and breeding:

The following study used tissue to enzyme weight ratios: 1 g: 2 mg. The digestion reaction was carried out under the conditions of a mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose (in the ratio of 3 ml of medium 0.3 g of tissue - i.e. 2 ml), containing the appropriate amount of collagenase II, i.e. 0.4 mg. The reaction was carried out for 14 hours. Finally, the cell pellet was resuspended in 1 ml of the mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose, containing 2% FBS and 2 mM glutamine for observation and qualitative evaluation (mainly evaluation of cell viability and condition after

After etching - e.g. regularity of shape was assessed) and quantitative etching reaction. A 92% viability of chondrocyte suspension was obtained, containing 0.42 million viable cells.

4) Combined culture - fragments of tissue with a suspension of chondrocytes

The cartilage tissue patches described in item 2 of this example, grown in the mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose, 2% FBS, 2 mM glutamine - were "flooded" with chondrocytes obtained by their growth after digestion ( service life in the range of 85% - 92%). An appropriate volume of the suspension containing 80,000 cells / 1.25 ml of medium was added to a well with the tissue slices. The supplement was then added, which in this study was chondroitin at a final concentration in the medium in the well of 0.01%.

The resulting tissue fragment and chondrocyte culture combination was incubated at 37 ° C, 5% CO 2 in a cell culture incubator for a total of 8 weeks until new tissue connections were established between the tissue fragments used. The process of "flooding" the tissue with a suspension containing each time a constant number of chondrocytes (80,000 cells per well, containing four tissue patches) was repeated once a week (every 7 days). Before the tissue was transferred and fresh medium and chondrocytes were added, the tissue slices were observed (as described in Example 4). The addition of chondrocytes took place after the then gentle transfer of tissue fragments into a new well of the 12-well plate.

After fourteen days of the test culture, the first visible changes in the cartilage tissue were noticed. After nineteen days, new tissue junctions between the old tissue fragments were identified as shown in Figure 5 - inverted microscopy image on the day of starting the culture (Figure 5A) and after seven days of cell-tissue culture (Figure 5B, C) using four pieces of tissue with a tangent section of 1 cm.

The new connections resembled the fibers that were formed between the patches of tissue, binding the patches. After seven weeks, the gaps between the old pieces of tissue were filled with new tissue and overgrown. The contact points of the two tissue patches were filled with new cellular connections. A new tissue flap was obtained, made of cartilage slices and free chondrocytes regularly supplied to the culture. A new tissue flap was obtained, approximately 3 mm x 1 cm x 0.8 cm in size.

Fig. 5 shows a microscopic image of the culture of the (first and second) tissue patches in medium 1 - visualization of the formation of new tissue connections over time. A: Image of fragments of two tissue slices (the first and the second) adhering to each other as closely as possible, on a tangent section of approx. 1 cm, on the first day of culture (photo documenting the start of culture). There is a gap between the first tissue patch closest to the edge of the plate and the next, second tissue patch. The first piece was gently trimmed at the edge of the tangent surface in order to identify it later in the experiment (the first piece fragment is at the bottom of the photo). B, C: Image of fragments of two tissue patches (first and second) and visible tissue connections between the contact surfaces, approx. 1 cm long, of two tissue patches on the twenty-seventh day of culture in medium 1 with the addition of 4.5 g / l glucose, 2% FBS, 2 mM glutamine and chondroitin at a concentration of 0.01%. Fiber-shaped joints are formed between the contact surfaces of the tissue patches. In the following days of culture, a new tissue will form between them, permanently bonding two tissue slices. Connections of the same type were also observed between the second and third patches, and the third and fourth patches, respectively. Pictures [A, B] taken with the 10x zoom, photo [C] with the 40x zoom.

Example 7 - an embodiment of the invention

Cultivation is performed as described in Example 5, except that:

1) Obtaining pieces of cartilage for further cultivation:

Five approximately rectangular tissue slices were obtained. The obtained slices were thin to a thickness of about 2 mm. They are 0.5 cm long and 0.4 cm wide.

2) Initial culture of the tissue fragments only:

Selected and appropriately cut slices as previously described were transferred with forceps to the wells of a 6-well sterile cell culture plate. Three selected slices of tissue approx. 2 mm thick and approx. 0.5 cm long and approx. 0.4 cm wide (contact surface length) were placed in a well previously filled with 2.5 ml of cell culture medium 2 with 4% serum FBS and 2 mM glutamine. The above tissue slices were oval in shape, two of which were

Claims (11)

Of the three patches, there was a convex contact surface and one slice had two concave surfaces. All the contact surfaces were compatible with each other, i.e. they adhered to each other very closely and as much as possible. In the well of the 6-well culture plate used, three slices of tissue were placed, arranged so that one of the edges (tangent edge) approx. 0.4 cm long adjoined each other. 3) Obtaining a chondrocyte suspension and breeding: The following study used tissue to enzyme weight ratios: 1 g: 4 mg. The digestion reaction was carried out under the conditions of a mixture of medium 1 and medium 2 with the addition of 4.5 g / l glucose (in the ratio of 6 ml of medium 0.3 g of tissue - i.e. 4 ml), containing the appropriate amount of collagenase II, i.e. 0.8 mg. The reaction was carried out for 8 hours. Finally, the cell pellet was resuspended in 1 ml of medium 2 with the addition of 4.5 g / l glucose, containing 4% FBS and 2 mM glutamine for observation and qualitative assessment (mainly assessment of the viability and condition of cells after digestion - e.g. regularity of shape was assessed) and quantitative digestive reaction. A chondrocyte suspension with 86% viability was obtained, containing 0.32 million viable cells. 4) Combined culture - fragments of tissue with a suspension of chondrocytes The cartilage tissue patches described in point 2 of this example, grown in medium 2 with the addition of 4.5 g / l glucose, 4% FBS, 2 mM glutamine - were "flooded" with chondrocytes obtained by growing them after digestion (viability in the range of 85 % - 92%). An appropriate volume of the suspension containing 60,000 cells was added to a well with tissue slices. The supplement was then added, which in this study was insulin at a final concentration in the medium in the well of 0.2%. The resulting tissue fragment and chondrocyte culture combination was incubated at 37 ° C, 5% CO 2 in a cell culture incubator for a total of 11 weeks until new tissue connections were established between the tissue fragments used. The process of "flooding" the tissue with a suspension containing each time a constant number of chondrocytes (60,000 cells per well, containing three tissue patches) was repeated once a week (every 7 days). Before the tissue was transferred and fresh medium and chondrocytes were added, the tissue slices were observed (as described in Example 4). The addition of chondrocytes took place after the then gentle transfer of tissue fragments into a new well of a 6-well plate. After twenty-one days of test culture, the first visible changes in the cartilage tissue were noticed. After forty-three days, new tissue connections were identified between the old pieces of tissue between all the tissue patches, that is, between the first and second, and the second and third. After eleven weeks, the gaps between the old pieces of tissue were filled with new tissue and overgrown. The contact points of the three tissue patches were filled with new cell connections. A new tissue flap was obtained, made of cartilage slices and free chondrocytes regularly supplied to the culture. A new tissue flap was obtained, approximately 2 mm x 1.5 cm x 0.4 cm in size. Patent claims A method of carrying out in vitro chondrocyte cell culture for obtaining material for the treatment of articular cartilage defects, characterized in that the healthy vitreous cartilage tissue collected from the patient is cut into at least two fragments of tissue from places with little traffic, and then these fragments are placed on the culture plate next to each other in the culture medium containing at least 2 mM glutamine, at least 2% FBS, at least 4.5 g / l glucose, with the shape of the tissue fragments being adjusted at least on one segment of the edge of each fragment so that, after placing the fragments close to each other, space between two similar tissue fragments is as small as possible and so that the space of the culture plate filled with tissue fragments is as small as possible in such a way that the fragments are joined together during the cultivation on at least one tangential segment, i.e. the tangential segment between at least two joined tissue fragments is at least 0.1 cm, with the cultivation up to chondrocyte suspensions are dispensed, and the cell culture of the chondrocyte tissue fragments is carried out, and the culture medium is changed at a predetermined time by adding chondrocytes to the culture medium, the cultures being continued until the tissue fragment is joined on at least a portion of the tangential segment. PL 240 714 B1 2. The method according to p. The process of claim 1, wherein the culture medium comprises supplements in a concentration of at least 0.01% IGF and / or 0.01% TGF and / or 0.01% chondroitin and or / or at least 0.1% insulin. 3. The method according to p. The method of claim 1 or 2, characterized in that the culture medium contains 55 - 75 mg / l sodium pyruvate and sodium bicarbonate, 2 mM - 4 mM glutamine, 2% - 10% FBS, and at least 4.5 g / l glucose, as well as ethanolamine and / or glutathione and / or ascorbic acid and / or insulin and / or transferrin and / or copper sulfate and / or manganese chloride. 4. The method according to p. A process as claimed in any one of the preceding claims, characterized in that prior to the addition of chondrocytes, the tissue fragments alone are cultured in medium, preferably for at least 14 days, after which the chondrocytes are added to the culture. 5. The method according to p. The method of claim 1 or 4, characterized in that the chondrocyte suspensions are added to the culture of the tissue fragments in such an amount that there are at least 40,000 chondrocytes per 2.5 ml of the culture medium. 6. The method according to p. The method of claim 1 or 4 or 5, characterized in that the minimum chondrocyte concentrations for culturing two fragments with a tangential segment of 0.1 cm-1 cm are 40,000 chondrocytes per 2.5 ml of medium. 7. The method according to p. The process of claim 1, wherein the chondrocytes to be cultivated are obtained by enzymatic digestion, and then the required number of cells is obtained by culturing the chondrocytes in a monolayer in adherent culture. 8. The method according to p. The process of claim 1, wherein the culturing is carried out at an elevated temperature of 37 ° C and 5% CO 2. 9. A method according to any one of claims 1 to 9 The method according to any of the claims 1-8, characterized in that the tangent segment between the at least two tissue pieces to be joined is at least 0.5 cm. 10. A method according to any one of claims 1 to 10 A method according to any of the claims 1-9, characterized in that the tissue fragments are approximately oval in shape. 11. The method according to any one of claims 1 to 11 A method according to any of the claims 1-10, characterized in that the shape of the tissue fragments is selected so that the tissue fragments in the culture adhere very closely to each other in the tangential segment.